Project Summary Neuronal microtubules are intimately interconnected with neuronal health, yet many basic principles that control neuronal microtubule organization remain mysterious. For example, microtubules can be nucleated throughout axons and dendrites, but regulators that position nucleation sites far from the cell body have not been identified. Nucleation is upregulated throughout the neuron by axon injury and stress so it is particularly important to understand how it is controlled. If basic mechanisms that control neuronal microtubule organization are not elucidated, it will be very difficult to understand the relationships between the microtubule regulatory proteins and neurodegeneration. Hereditary spastic paraplegia is genetically one of the simplest forms of neurodegenerative disease, and mutations in the gene that encodes the microtubule severing protein spastin cause 40% of cases. However, how and where spastin functions in mature neurons has not been pinned down, and why its reduction sensitizes neurons to degeneration is also not clear. In this proposal Drosophila is used as a model system in which to efficiently identify basic mechanisms that regulate neuronal microtubules to provide a framework for future work on regeneration and degeneration. Aim 1. An unexpected pathway controls microtubule nucleation in dendrites. Using a screening approach, Wnt signaling proteins emerged as key regulators that position microtubule nucleation sites in dendrites. Preliminary data in this aim indicates Wnt signaling proteins likely activate nucleation on endosomes. These preliminary findings will be strengthened by pairing a new nucleation assay with localization of Wnt signaling proteins and endosomes. Aim 2. The role of severing proteins in converting minus end nucleation to minus end growth. It was recently shown that minus ends grow in dendrites and this is important for polarity control and dendrite regeneration. Preliminary data indicates spastin plays a role in generating growing minus ends. Using new assays to visualize nucleation and severing in combination with genetic tools, how and where microtubules are severed in dendrites will be investigated. Aim 3. Control of disruptive nucleation by a checkpoint system. Microtubule nucleation has the potential to disrupt polarity by generating microtubules in random orientations. Detailed live imaging of growing microtubule plus ends determined that a checkpoint system depolymerizes microtubules nucleated in the ?wrong? orientation. Two players required for promoting growth of ?right? orientation microtubules have been identified. Their function, localization and interactions with one another will be investigated. Conclusion: We will elucidate fundamental but poorly understood mechanisms that control dendrite microtubule organization to fill in key gaps in our understanding of the neuronal cytoskeleton that are essential context for understanding neuronal regeneration and degeneration.